WO2017003079A1

WO2017003079A1 - Oblique incidence, prism-incident, silicon-based, immersion microchannel-based measurement device and measurement method

Info

Publication number: WO2017003079A1
Application number: PCT/KR2016/004386
Authority: WO
Inventors: 조현모; 제갈원; 조용재
Original assignee: 한국표준과학연구원
Priority date: 2015-06-30
Filing date: 2016-04-27
Publication date: 2017-01-05
Also published as: US20180100795A1; SE1750873A1; KR101684138B1; SE541939C2; US10921241B2; JP2018503810A; JP6483834B2

Abstract

An oblique incidence, prism-incident, silicon-based, immersion microchannel-based measurement device related to one example of the present invention may comprise: a microchannel structure having a support, a substrate formed, from a semiconductor or a dielectric material, on the support, a cover part having a prism structure and installed on the support, and a microchannel formed in the upper portion of the support or the lower portion of the cover part; a sample injection part for injecting a buffer solution containing a sample of a biomaterial into the microchannel so as to form, on the substrate, an adsorption layer of the sample; a polarized light generation part which radiates, through an incident surface of the prism, polarized incident light onto the adsorption layer at an incident angle meeting a P-wave antireflection condition; and a polarized light detection part to which first reflected light, reflected from the adsorption layer and/or the substrate, is incident, having passed through a reflective surface of the prism, and which detects the change in polarization of the first reflected light.

Description

Device and method for measuring immersion structure prism incident silicon-based immersion microchannel

This study relates to the inclination structure of molecular bonding properties of biomaterials and the refractive index of buffer solution under the immersion microfluidic environment. More specifically, by measuring the bottom of the prism and the substrate attached to the trapezoidal micro-channel inclined to effectively remove the light reflected from the interface between the prism and the medium, and to minimize the scattering of the micro-channel interface and the measuring device capable of high sensitivity measurement method using the same It is about.

Reflectometry and ellipsometry are optical analysis techniques that determine the thickness or optical properties of a sample by measuring the change in reflectance or polarization state of the reflected light reflected from the surface of the sample and analyzing the measured values.

As measurement equipment using this, there is a reflectometer and an ellipsometer. They are used to evaluate various thin film thickness and physical properties of nano thin film manufacturing process in semiconductor industry. In addition, efforts are being made to extend the scope of application to the bio industry and apply it to interface analysis of bio materials such as proteins, DNA, viruses, and new drugs.

Conventional reflectometers are sufficient to evaluate the thickness and physical properties of nano-films with dimensions of several nanometers (nm) or more, but have low sensitivity in analyzing low molecular weight biomaterials that require sensitivity in the range of approximately 1 to 0.001 nanometers. There is a problem that the reliability is lowered. The ellipsometer has a measurement sensitivity of less than 0.01 nm compared to the reflectance measuring instrument. In particular, the measurement sensitivity is high under conditions where the refractive index is large, such as the thickness of an oxide film having a smaller refractive index than that of a semiconductor on a high refractive index semiconductor substrate.

However, in the case of an ellipsometer, a measurement method with improved sensitivity is required to analyze even low molecular weight biomaterials.

As a conventional technique for improving measurement sensitivity when analyzing a biomaterial, a surface plasmon resonance sensor (hereinafter referred to as an 'SPR sensor') in which a reflectance measuring method and a surface plasmon resonance (SPR) technology is mixed have.

Surface plasmon resonance (SPR) is a phenomenon in which electrons present on a metal surface are excited by light waves, causing collective vibration in the longitudinal direction of the surface, where light energy is absorbed. Say SPR sensor can measure the change of thickness and refractive index of nano thin film in contact with metal surface by using surface plasmon resonance which is sensitive to the polarization property of light. It is known that non-labeling can be measured in real time.

SPR sensor uses the principle that the structure of the glass is coated with a metal thin film of tens of nanometers on the material such as glass and the biomaterial can be bonded on it, and the resonance angle changes when the sample dissolved in the buffer solution is bonded to the sensor. The resonance angle is achieved by measuring the reflectance. When light enters the SPR sensor, the glass material becomes the incident medium and passes through the thin film layer to which the biomaterial is bonded. Finally, the buffer solution corresponds to the substrate.

In such a structure, the refractive index of the complete solution corresponding to the substrate material directly affects the movement of the resonance angle, similar to the change of the biological thin film layer due to the bonding of the sample to be measured. Therefore, in order to measure only pure bonding dynamics, the refractive index of the buffer solution should be measured and corrected independently.

In order to correct the refractive index change of the buffer solution and to prevent errors due to diffusion between the sample and the buffer solution, a sophisticated valve device, an air injection device, and a method of using one as a reference channel using two or more channels and correcting It is used. However, it is difficult to distinguish between the SPR angle change due to the refractive index change of the buffer solution and the SPR angle change due to the pure adsorption and dissociation characteristics, and it can always act as a measurement error factor. As a result, the conventional SPR sensor has a fundamental difficulty in measuring the adsorption and dissociation characteristics of a material having a low molecular weight, such as a low molecule, due to the limitation of the above measurement method.

In addition, the conventional SPR sensor uses a metal thin film of a noble metal such as gold (Au), silver (Ag) for the surface plasmon resonance is expensive manufacturing of the sensor. In addition, the metal thin film has an uneven surface roughness depending on the manufacturing process, and the variation in refractive index is severe, and because of unstable optical properties, it is difficult to quantitatively measure biomaterials and to detect errors due to different sensitivity characteristics at different positions when compared with reference channels. There is a problem to include.

To improve the shortcomings of the SPR sensor, a biomaterial junction sensor layer is formed on a substrate material such as silicon, and the ellipsometric method measures the amplitude and phase of the light reflected from the substrate material through a buffer solution under an immersion microenvironment under p-wave antireflection conditions. When measured by, it is possible to obtain a signal whose amplitude is not sensitive to the change in refractive index of the buffer solution and sensitive to the bonding dynamics of the biological material. In the case of measuring the bonding characteristics of the biomaterial adsorbed to the substrate material under the immersion microfluidic environment, the buffer solution becomes the incident medium and the light passing through the biomaterial adsorption layer is reflected from the substrate material as opposed to the SPR measurement.

Under these measurement conditions, the measured elliptic measurement angle is not sensitive to the change in the refractive index of the incident medium, which is a buffer solution, but only a change in the biofilm and the substrate material. In the case of a stable refractive index substrate such as silicon, the measured elliptic measurement angle Ψ is sensitive only to changes in the biofilm, and the elliptic measurement angle Δ represents a signal sensitive only to the refractive index of the buffer solution. Can be measured simultaneously. However, when using a substrate parallel to a planar incidence structure such as a prism, the light reflected from the interface between the prism and the buffer solution should be removed and only the light reflected from the substrate should be used. In order to minimize the amount of sample used, the distance between the surface of the prism and the substrate material should be reduced. In this case, the two reflecting lights are located at a very close distance, which makes it difficult to separate and cause a measurement error. Therefore, in a planar incident structure such as a prism, a new structure measuring method is required to distinguish the light reflected from the interface between the prism and the buffer solution and the light reflected from the substrate material including the sensor.

1 is a configuration of the prior patent to measure the sensor layer on the substrate material such as silicon in order to improve the shortcomings of the SPR sensor and measured by the ellipsometric method under the immersion microfluidic environment. As shown in FIG. 1, the biomaterial bonding characteristic sensor according to the prior patent has a microchannel structure 100, a substrate 120, a cover 140, a microchannel 150, a sample injection unit 200, It consists of a polarization generating unit 300 and a polarization detecting unit 400. In the biomaterial bonding characteristic sensor according to the prior patent, the adsorption layer 160 is placed on the substrate 120 or the dielectric thin film 130, and the liquid immersion microchannel 150 is formed. At this time, when the buffer solution 210 in which the sample 1 of the biomaterial is dissolved is injected into the micro channel 150, the biomaterial is adsorbed onto the ligand 2 formed on the surface of the adsorption layer 160. Thus, an adsorption layer having a predetermined thickness is formed.

The polarized incident light generated from the polarization generator 300 is incident on the interface between the buffer solution 210 and the substrate 120 at an angle causing p-wave antireflection conditions through the incident surface 142. In this case, the reflected light reflected from the substrate 120 includes optical data regarding the refractive index of the adsorption layer and the buffer solution of the sample 1. That is, in the process of adsorbing and dissociating the sample 1 to the ligand 2, the molecular adsorption and dissociation kinetics such as the adsorption concentration, the thickness or refractive index of the adsorption layer, and the refractive index of the buffer solution are changed, As a result, the measured elliptic measurement angles are changed. The reflected light including the optical data is detected by the polarization detector 400 via the buffer solution 210 and the reflective surface 144. In this case, the polarization detector 400 may determine the molecular adsorption and dissociation dynamics of the sample 1 and the refractive index of the buffer solution by measuring the change according to the polarization component of the reflected light, that is, the ellipsometric angles.

2 shows an adsorption curve showing a process in which the sample 32 is adsorbed to the metal thin film 20, and a dissociation curve showing a dissociation process. The larger the association rate constant (ka), the faster the absorption of the biomaterial, and the smaller the dissociation rate constant (kd), the slower the dissociation.

That is, the dissociation constant (KD = kd / ka) at equilibrium can be obtained by measuring the adsorption rate constant and dissociation rate constant. For example, it is possible to determine whether a low-molecular drug candidate that can be used as a carcinogen can be used as a new drug by measuring the adsorption or desorption property of a protein including a carcinogenic factor.

Hereinafter, the features and limitations of the biomaterial analysis sensor according to the preceding characteristics will be described with reference to FIGS. 3 and 4. When the light is incident using the prism incidence structure as shown in FIG. 3, the light is incident on the interface at an inclination angle of about θ ₂ = 70.85 °, and when the light is incident from the prism to the buffer solution by a change in refractive index of the buffer solution (0.0002). An angle change of 0.024 ° will appear. The p-wave antireflection condition is near θ ₂ = 70.85 °, but the current angle due to the change in the refractive index of the buffer solution is changed to 0.024 ° to 70.826 °, so as shown in FIG. 4, graphs of Ψ and △ are displayed and the refractive index changes. Since the p-wave antireflection angle hardly changes, the values of Ψ and Δ are measured at 70.826 °, a small angle of 0.24 °.

In FIG. 4, when the refractive indexes of the buffer solution 210 are different from each other, the solid line graph has a refractive index of 1.3330 and the dotted line graph corresponds to the refractive index of 1.3332 of the buffer solution 210. When the prism structure is used, the measurement result due to the change of the incident angle shows that the change of Ψ value acts in a direction that cancels the small change in the vertical incidence structure and hardly shows a change, whereas Δ shows a large change. That is, since the elliptic measurement constant Δ of the phase difference shows a sensitive change only in the refractive index change of the buffer solution and is hardly affected by the bonding property, only the change in the refractive index of the buffer solution can be measured with high sensitivity. The change in elliptic measurement constant △ shows a very large change as the thickness of the thin film material becomes very small, and when used in an applied study for analyzing the change in material properties or bonding properties by measuring the change in refractive index, It is a measuring method that can measure high sensitivity refractive index.

When a buffer solution having a different refractive index, such as a buffer solution continuously supplied and a solvent used in a sample, is supplied to a sensor through a microchannel, pure bonding dynamics and a change in the refractive index of the buffer solution can be simultaneously measured.

However, in FIG. 3, when the distance between the bottom of the prism and the substrate material is small, it is difficult to separate the light reflected from the interface between the prism and the buffer solution and the light reflected from the substrate material. Because the measurement under the p-wave antireflection conditions, the intensity of the light reflected from the substrate material is relatively weaker than the light reflected at the interface between the prism and the buffer solution may cause a measurement error.

The present invention is to solve the above problems, the measuring device and measuring method for separating the light reflected from the substrate material and the reflected light at the interface between the prism and the buffer solution when the distance between the bottom of the prism and the substrate material is small To provide. In particular, the present invention provides a measuring apparatus and a measuring method for solving the problem that measurement errors occur because the intensity of light reflected from the substrate material is relatively weaker than the light reflected at the interface between the prism and the buffer solution because it is measured under p-wave antireflection conditions. .

Other objects, specific advantages and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments in conjunction with the accompanying drawings.

An inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring apparatus according to an example of the present invention for realizing the above object is provided with a substrate or a prism structure composed of a semiconductor or a dielectric formed on a support and a support on the support. A micro channel structure having a cover part to be installed and a micro channel formed at any one of the upper part of the support and the lower part of the cover part; A sample injection unit injecting a buffer solution containing a sample of a biomaterial into a micro channel to form an adsorption layer of the sample on a substrate; A polarization generator for irradiating incident light polarized through the incident surface of the prism to the adsorption layer at an incident angle satisfying a p-wave antireflection condition; And a polarization detector configured to receive first reflected light reflected from at least one of the adsorption layer and the substrate through the reflective surface of the prism, and detect a change in polarization of the first reflected light. It may be formed to form a predetermined inclination angle with the bottom surface of the prism.

In addition, the first reflected light travels in a different direction from the light reflected from the bottom of the prism.

The polarization detection unit may separate and detect the first reflected light and the light reflected from the bottom of the prism.

In addition, an opening portion may be formed on an upper surface of the support.

In addition, the through part may have a trapezoidal shape, and the through part may have a trapezoidal shape such that an upper side of the penetrating part has a length smaller than a lower side of the penetrating surface.

In addition, the incident light may be irradiated onto the adsorption layer through the opened through part, and the trapezoidal shape may block reflection of a portion of the incident light.

In addition, the upper side of the trapezoidal shape forms a first inclined portion inclined upper side, the lower side of the trapezoidal shape forms a second inclined portion inclined upper side.

Further, cross sections of the first inclined portion and the second inclined portion become narrower toward the tip, and the tip of the first inclined portion is located below the tip of the second inclined portion.

In addition, an inflow path through which the buffer solution flows into the microchannel is formed on an upper side of the trapezoidal upper side of the penetrating part, and a discharge path through which the buffer solution is discharged into the microfluid flower introduced below the trapezoidal lower side of the penetrating part. Can be formed.

In addition, the angle of inclination has a range between 0 seconds and 10 degrees.

In addition, the through part may be formed in a trapezoidal shape, and the trapezoidal shape of the through part may be formed such that an upper side of the penetrating surface has a length greater than a lower side of the penetrating surface.

The microchannel structure may further include a dielectric thin film provided between the substrate and the absorption layer, and the first reflected light may further include light reflected from the dielectric thin film.

On the other hand, the liquid immersion microfluidic measuring device according to an embodiment of the present invention for realizing the above-described object is a substrate composed of a semiconductor or a dielectric formed on a support and a support, a cover portion provided with a flat plate structure and installed on the support and the A micro channel structure having a micro channel formed on one of an upper portion of the support and a lower portion of the cover part; A sample injection unit injecting a buffer solution containing a sample of a biomaterial into a micro channel to form an adsorption layer of the sample on a substrate; A polarization generator for irradiating incident light polarized through the plane of incidence of the flat plate to the adsorption layer at an incidence angle satisfying a p-wave antireflection condition; And a polarization detector configured to receive first reflected light reflected from at least one of the adsorption layer and the substrate through the reflective surface of the flat plate, and detect a change in polarization of the first reflected light. It may be formed to form a predetermined inclination angle with the bottom of the plate.

On the other hand, the microfluidic structure according to an example of the present invention for realizing the above object, the support; A substrate composed of a semiconductor or dielectric formed on a support; A cover part provided in a prism structure and installed on the support; And a microchannel formed at any one of the upper portion of the support and the lower portion of the lid, wherein a buffer solution containing a sample of a biomaterial is injected into the microchannel to form an adsorption layer of the sample on the substrate. The incident light polarized through the incidence plane of the prism is irradiated onto the adsorption layer at an incidence angle satisfying the p-wave antireflection condition, and the first reflected light reflected from at least one of the adsorption layer and the substrate is reflected through the reflection plane of the prism. It is emitted, the surface of the substrate may be formed to form a predetermined inclination angle with the bottom surface of the prism.

On the other hand, the microfluidic structure according to an example of the present invention for realizing the above object, the support; A substrate composed of a semiconductor or dielectric formed on a support; A cover part provided in a flat structure and installed on the support; And a microchannel formed at any one of the upper portion of the support and the lower portion of the lid, wherein a buffer solution containing a sample of a biomaterial is injected into the microchannel to form an adsorption layer of the sample on the substrate. The incident light polarized through the plane of incidence of the plate is irradiated onto the adsorption layer at an incidence angle that satisfies the p-wave antireflection condition, and the first reflected light reflected from at least one of the adsorption layer and the substrate passes through the plane of reflection of the plate. It is emitted, the surface of the substrate may be formed to form a predetermined inclination angle with the bottom surface of the plate.

On the other hand, the liquid immersion microchannel measurement method according to an example of the present invention for realizing the above object, the first step of injecting a buffer containing a sample of the biomaterial into the microchannel of the sample flow path microchannel structure; A second step of adsorbing the sample onto the substrate of the microchannel structure to form an adsorption layer; A third step of polarizing the light polarizing unit incident light on the adsorption layer through an incident surface of the prism of the microchannel structure at an incident angle satisfying the p-wave antireflection condition; A fourth step in which first reflected light reflected from at least one of the adsorption layer and the substrate is incident through a reflecting surface of the prism; And a fifth step of detecting a polarization change of the first reflected light by the polarization detector, wherein the surface of the substrate may be formed to have a predetermined inclination angle with a bottom surface of the prism.

On the other hand, the liquid immersion microchannel measurement method according to an example of the present invention for realizing the above object, the first step of injecting a buffer containing a sample of the biomaterial into the microchannel of the sample flow path microchannel structure; A second step of adsorbing the sample onto the substrate of the microchannel structure to form an adsorption layer; A third step of polarizing the polarizer generating light to be incident on the adsorption layer at an incident angle satisfying a p-wave antireflection condition through an incident surface of the flat plate of the microchannel structure; A fourth step in which first reflected light reflected from at least one of the adsorption layer and the substrate is incident through a reflecting surface of the plate; And a fifth step of detecting a polarization change of the first reflected light by the polarization detector, wherein the surface of the substrate may be formed to have a predetermined inclination angle with a bottom surface of the flat plate.

The first reflected light may travel in a different direction from the light reflected from the bottom of the prism. In the fifth step, the polarization detector may separate and detect the first reflected light and the light reflected from the bottom of the prism. have.

In addition, the fifth step may include polarizing the first reflected light by an analyzer; Detecting the polarized first reflected light by a photodetector to obtain predetermined optical data; And an analysis means obtains an elliptic measurement constant relating to the phase difference of the elliptic measurement method based on the optical data, obtains the refractive index of the buffer solution, obtains an elliptic measurement constant relating to the amplitude ratio, and includes a measurement including adsorption concentration, adsorption and dissociation constant of the sample. Deriving a value; may further include.

As described above, the inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring apparatus and measuring method according to the present invention incline the substrate attached to the bottom of the prism and the microfluidic, so that the sensor is attached to the interface between the prism and the medium. In contrast to the propagation direction of the scattered light reflected from the flow path structure, only the signal reflected from the substrate material is separated and detected, thereby obtaining a high sensitivity measurement sensitivity. In the conventional measuring method, the light reflected from the interface between the prism and the measuring medium has a larger energy than the light reflected from the substrate material and is difficult to separate, which may cause a measurement error. Although there was a problem that the measurement range that can be measured for is limited, even when manufactured by inclining an angle of about 2 degrees, there is an advantage that the light reflected from the interface between the prism and the measurement medium and the light reflected from the substrate material can be sufficiently separated.

In particular, by using a trapezoidal microchannel structure and injecting light into a narrow microchannel structure and allowing light to travel through the wide microchannel structure, the narrow microchannel structure acts as a preliminary aperture, There is an advantage to minimize the double scattering. In addition, a multi-channel micro-channel can be manufactured and light can be incident on a large area using a cylinder lens or the like to measure a simple and highly sensitive multi-channel micro-channel.

In addition, the microchannel structure of the present invention has a trapezoidal microchannel coupled with a prism structure optimized for analysis of biomaterials, and consists of a single channel formed with a multichannel or a plurality of self-assembled monolayer films. Therefore, it is possible to provide various types of experimental conditions, such as varying the concentration of the sample into the multi-channel microchannel or injecting the self-assembled monolayer membrane, thereby increasing the efficiency in the analysis of biomaterials. have.

In addition, the present invention can be used in a variety of industries, such as bio, medical, food, environment can be measured highly sensitive to the biomaterial in a non-labeled manner in the immersion micro-channel environment.

Although the present invention has been described in connection with the above-mentioned preferred embodiments, it will be readily apparent to those skilled in the art that various other modifications and variations are possible without departing from the spirit and scope of the present invention. Are all within the scope of the appended claims.

The following drawings, which are attached to this specification, illustrate one preferred embodiment of the present invention, and together with the detailed description thereof, serve to further understand the technical spirit of the present invention. It should not be construed as limited.

1 is a cross-sectional view showing a biomaterial bonding characteristics measuring sensor according to the prior patent,

Figure 2 is a schematic diagram showing the adsorption concentration change in the process of the sample is adsorbed, dissociated to the metal thin film,

Figure 3 is a schematic diagram for explaining the problems of the prior art in which the inherent adsorption and dissociation dynamics of the sample appearing through the adsorption, dissociation process of the sample and the change of the refractive index by the buffer solution is mixed,

4 is a graph measuring elliptic measurement constants Ψ and △ by the change of the refractive index of the biomaterial adsorption and buffer solution using a biomaterial bonding characteristic measurement sensor of the prior patent,

5 is a cross-sectional view showing the configuration of an inclined incident structure prism incident type silicon-based liquid immersion microchannel measuring apparatus according to an embodiment of the present invention;

Figure 6a is a perspective view of a multi-channel microchannel structure in accordance with an embodiment of the present invention,

6B is an exploded perspective view of a multichannel microchannel structure according to an embodiment of the present invention;

7 is a perspective view of a multichannel microchannel structure according to another embodiment of the present invention;

8A is a perspective view of a support according to an embodiment of the present invention;

8B is a bottom perspective view of a support according to an embodiment of the present invention;

9 is a cross-sectional view of the support according to an embodiment of the present invention,

10 is a flow chart of a method of measuring the immersion microfluidic channel according to an embodiment of the present invention;

Figure 11 is a schematic diagram showing the path of the light reflected from the sample in the prism vertical incidence structure of the prior patent,

12A is a schematic diagram showing a path of light reflected by an immersion microfluidic measuring device according to an embodiment of the present invention;

12B is a method of measuring a trapezoidal microfluidic structure and an oblique incidence structure prism-incident type silicon-based liquid immersion microchannel according to an embodiment of the present invention;

Figure 13a is a schematic diagram showing the path of the light reflected from the immersion microfluidic measuring apparatus according to another embodiment of the present invention,

FIG. 13B illustrates a structure of a trapezoidal microchannel and an oblique incidence structure prism-incident type silicon-based immersion microchannel measurement method according to another exemplary embodiment of the present invention.

First, with reference to the accompanying drawings looks at the configuration of the inclined incident structure prism incident type silicon-based immersion microfluidic measuring apparatus according to an embodiment of the present invention.

Figure 5 is a schematic diagram showing the configuration of an inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring apparatus according to an embodiment of the present invention. As shown in FIG. 5, an apparatus for simultaneously measuring molecular bonding characteristics and a buffer solution refractive index according to an exemplary embodiment of the present invention provides an incident light with the microfluidic structure 100 and the sample injector 200 that provide a large immersion microchannel environment. The optical system includes a polarization generator 300 and a polarization detector 400 for detecting a change in polarization of reflected light.

The present invention is to measure the adsorption and dissociation dynamics of biomaterials, including low molecular weight using an ellipsometric method, a buffer (210) containing a sample (not shown) of the biomaterial in the sample injection unit 200 It has a structure which is injected into this micro flow path structure 100. At this time, the micro-channel structure 100, as described below, the micro-channel 150 is composed of a multi-channel or a single channel.

6A is a perspective view illustrating an example of a multichannel microchannel structure according to the present invention, and FIG. 6B is an exploded perspective view of the multichannel microchannel structure. As shown in FIGS. 6A and 6B, the microchannel structure 100 includes a support 110, a substrate 120, an adsorption layer 160, and a cover 140, and a plurality of microchannels 150. Is formed to form a multi-channel structure.

The support 110 has a rectangular plate shape as shown in FIG. 6B, and a groove 112 for forming the substrate 120 and the adsorption layer 160 is formed. In addition, the inflow path 152 and the discharge path 154 of the micro flow path 150 are formed at one side and the other side around the groove 112. In this case, the groove 112, the inflow path 152 and the discharge path 154 are formed using a semiconductor etching technique or an exposure technique.

The substrate 120 is formed in the groove 112 of the support 110 in the form of a square plate. According to an embodiment of the present invention, the substrate 120 uses silicon (Si) having a complex refractive index of about 3.8391 + i0.018186 at 655 nm and providing constant and stable physical properties at low cost. However, the material of the substrate 120 may be a semiconductor or dielectric material other than silicon.

The adsorption layer 160 serves to absorb and dissociate a sample of a low molecular weight biomaterial and reflect incident light. The surface junction 160 is formed on the upper side of the substrate 120, as shown in Figure 5 and 6b.

Adsorption layer 160 according to an embodiment of the present invention may be composed of at least one of a self-assembled thin film and a bio thin film. In addition, in an embodiment of the present invention, the dielectric thin film 130 may be further included between the silicon substrate 120 and the adsorption layer 160.

The dielectric thin film 130 is a thin film material of a transparent material including a semiconductor oxide film or a glass film is used. The thickness of the dielectric thin film is preferably 0 to 1000 nm. On the other hand, an example of the dielectric thin film 130 that can be easily obtained is a silicon oxide film (SiO 2) grown to several nanometers by naturally oxidizing silicon. Since the refractive index of the silicon oxide film is about 1.456 at 655 nm, the difference in refractive index between the silicon oxide film and the substrate 120 made of silicon is large, which helps to increase the measurement sensitivity of the present invention.

In addition, a glass film made of optical glass may be used for the dielectric thin film 130. The dielectric thin film 130 made of silicon, silicon oxide film, or glass film can provide a stable refractive index compared to a metal thin film such as gold or silver, thereby providing stable optical characteristics and lowering manufacturing costs. .

The cover part 140 may be installed on the support 110 as shown in FIGS. 5 to 6B, and may include a prism 142 and a partition wall 146. The light incident on the incident surface 143 of the prism 142 causes the microfluidic medium to be refracted by the microfluidic structure combined with the prism 142. The refracted light is angled to satisfy the p-wave antireflection condition on the substrate material. To be incident.

In addition, the cover part 140 is provided with a plurality of partitions 146 for forming the micro-channel micro-channel 150 as the lower surface of the prism 142 as shown in FIG. 6B. The prism 142 and the fine flow path structure may be made of a transparent material such as glass or transparent synthetic resin material, but for ease of fabrication, the whole including the fine flow path partition wall 146 may be integrally formed using a molding method or the like. Can be.

Meanwhile, as an example of the synthetic resin material, an acrylic resin such as polymethyl methacrylate (PMMA) may be used. And silicon-based materials such as silicon phosphate polymer (PDMS, polydimethylsiloxane) may also be used.

The micro-channel 150 is a plurality of passages through which the buffer solution 210 including the sample is introduced or discharged. That is, as described above, each space between the partition walls 146 of the cover part 140 communicates with the inflow passage 152 and the discharge passage 154 formed in the support 110 so that a plurality of the microfluidic structures 100 may be formed. The micro channel 150 is formed. At this time, the width of the micro channel 150 has a micro scale of about several mm or less than 1 mm.

7 is a perspective view showing another example of the multi-channel microchannel structure according to the present invention. As shown in FIG. 7, the cross-section of the prism 142 may have a trapezoidal shape in the multichannel microchannel structure 100. In this case, the polarization generating unit 300 and the polarization detecting unit 400 illustrated in FIG. 5 may cause incident light and reflected light to the incident surface 143 and the reflective surface 144 of the prism 142 to be vertical or polarized. It is fixed at a position where it is incident or close to perpendicular at an angle that does not change. If a flat plate is used instead of a prism structure, there is a loss of incident light. However, a flat plate structure may be used for a simple structure.

Looking at the structure of the support 110 is applied to the present invention. Figure 8a is a perspective view of the support according to an embodiment of the present invention, Figure 8b is a bottom perspective view of the support according to an embodiment of the present invention, Figure 9 is a cross-sectional view of the support according to an embodiment of the present invention.

An open through portion 116 is formed on the upper surface of the support 110, and the through portion 116 is opened in a trapezoidal shape. The trapezoidal shape of the through part 116 is formed so that the upper side located in the incident surface direction has a length smaller than the lower side located in the reflective surface direction. Accordingly, the through part 116 may act as an aperture to prevent scattering due to the micro channel structure.

As shown in FIG. 9, the upper side of the trapezoidal shape of the penetrating portion 116 forms a first inclined portion 114 having an inclined upper side, and the lower side of the trapezoidal shape of the penetrating portion 116 is inclined upper side. The two inclined portion 115 is formed. The second inclined portion 115 is inclined in a direction opposite to the first inclined portion 114.

In FIG. 9, the angle formed by the first inclination part 114 and the second inclination part 115 is represented by 10 °, but the present invention is not limited thereto and may be designed at an angle within a range of approximately 10 ° to 80 °. Can be. The inclination of the first inclined portion 114 and the second inclined portion 115 smoothly induces the flow of the micro-channel 150. The buffer solution 210 introduced into the microchannel 150 through the inflow path 152 may proceed smoothly along the inclination of the first inclination part 114, and the discharge path along the inclination of the second inclination part 115. You may proceed smoothly to 154.

Cross sections of the first inclination portion 114 and the second inclination portion 115 become narrower toward the tip, and the tip of the first inclination portion 114 is located below the tip of the second inclination portion 115. do. The angle between the line segment formed on the upper surface of the support 110 and the line segment connecting the tip of the first inclined portion 114 and the tip of the second inclined portion 115 is preferably set to about 2 °.

The present invention may be implemented as a single channel microchannel structure. The microchannel structure 100 of one channel includes one microchannel 150. That is, the cover part 140 includes a prism 142 and a pair of partition walls 146 formed at both ends of the lower surface of the prism, and the support 110 has one inflow path 152 and a discharge path 154. As a result, the microchannel 150 of the single channel is formed.

Then, a different adsorption layer is formed on a plurality of different self assembled monolayer (SAM) 132 or the same self assembled monolayer film. The self-assembled monolayer film 132 is formed by spontaneously arranging monomers formed of a head and a tail group by a chemical adsorption of molecules. In this case, the interface characteristics of the self-assembled tomography films 132 may be changed by chemically converting the functional groups of the tail groups of the self-assembled tomography films 132. That is, each of the self-assembled monolayer films 132 has a sensor structure that varies the degree of adsorption and dissociation with the sample, and can simultaneously measure various adsorption and dissociation dynamics of the biomaterial.

As illustrated in FIG. 5, the sample injector 200 injects a buffer solution 210 including a sample (not shown) of a low molecular weight biomaterial into the inflow path 152 of the microchannel 150. The sample injecting unit 200 has a structure for dissolving the sample in a predetermined concentration in the buffer solution 210, a valve device (not shown) that can inject and block the buffer solution 210 in the micro-channel (150) Equipped.

In this case, the sample injection unit 200 may inject the buffer solution 210 into the micro-channel 150 of each channel by varying the concentration of the sample or by placing a time difference. Meanwhile, when the buffer solution 210 is injected into the micro channel 150, a portion of the sample (not shown) is adsorbed on the dielectric thin film 130 to form an adsorption layer 160 having a predetermined thickness. In this case, the adsorption layer 160 may be a multilayer film composed of various biomaterials including a self-assembled monolayer film 132 suitable for bonding characteristics of various biomaterials, an immobilization material, and low molecules bonded to the immobilization material.

As shown in FIG. 5, the polarization generator 300 irradiates the absorption layer 160 with incident light polarized through the incident surface 143 of the prism 142 of the micro-channel structure 100. The polarization generator 300 may include a light source 310, a polarizer 320, and other collimating lenses 330, a focusing lens 340, or a first compensator 350 as essential components. have.

In this case, the polarizer 320 and the first compensator 350 may be configured to be rotatable or may further include other polarization modulating means. On the other hand, the polarized incident light has a polarization component of p-wave and s-wave, and in order to increase the signal-to-noise ratio, light of nearly p-wave can be incident. At this time, in the present invention, the incident light should be irradiated at an incident angle θ that satisfies the p-wave antireflection condition. In the elliptic measurement equation, the complex reflection coefficient ratio (ρ) can be expressed as the ratio of the reflection coefficient ratio (Rp) of the p wave to the reflection coefficient ratio (Rs) of the s wave, that is, ρ = Rp / Rs. Denotes a condition that the reflection coefficient ratio Rp of the p-wave is close to zero. The p-wave antireflection condition is similar to the surface plasmon resonance condition of the conventional SPR sensor, and is a condition in which the measurement sensitivity of the present invention is maximized.

The light source 310 irradiates monochromatic light in the infrared, visible or ultraviolet wavelength range, or irradiates white light. As the light source 310, various lamps, light emitting diodes (LEDs), lasers, laser diodes (LDs), and the like may be used. In this case, the light source 310 may have a structure capable of varying the wavelength according to the structure of the optical system. On the other hand, in the vicinity of the above-described p-wave anti-reflective conditions, the magnitude of the optical signal of the reflected light may be relatively small. In this case, a high sensitivity is generated by irradiating light with a high amount of light using a laser or a laser diode (LD) to increase the signal-to-noise ratio. Measurements can be made possible.

The polarizer 320 is provided with a polarizing plate to polarize the light emitted from the light source 310. At this time, the polarization component has an s wave in a direction perpendicular to a p wave in a direction parallel to the incident surface.

The collimating lens 330 receives light from the light source 310 to provide parallel light to the polarizer 320. In addition, the focusing lens 340 may converge parallel light passing through the polarizer 320 to increase the amount of incident light. In addition, the first compensator 350 serves to retard the polarization component of the incident light.

As illustrated in FIG. 5, the polarization detector 400 receives the reflected light reflected from the adsorption layer 160 through the reflective surface 144 of the prism 142 and detects a change in the polarization state. The polarization detector 400 includes an analyzer 410, a detector 420, and an operation processor 430 as essential components, and a second compensator 440 and a spectrometer 450. It may be provided. In this case, the analyzer 410 corresponds to the polarizer 320 and may include a polarizing plate to polarize the reflected light again to control the degree of polarization of the reflected light or the direction of the polarization plane. In addition, the analyzer 410 may be configured to be rotatable according to the structure of the optical system, or may further include polarization modulating means capable of performing a function such as phase change and cancellation of the polarization component.

The photodetector 420 detects polarized reflected light to obtain optical data, and converts it into an electrical signal. At this time, the optical data includes information on the change of the polarization state in the reflected light. The photodetector 420 may be a CCD solid-state image pickup device, a photomultiplier tube (PMT), or a silicon photodiode.

The operation processor 430 serves to derive a measurement value by obtaining an electrical signal from the photodetector 420. The arithmetic processor 430 includes a predetermined analysis program using a reflectance measuring method and an elliptic measurement method. The arithmetic processor 430 extracts and analyzes optical data converted into an electrical signal, so that the adsorption concentration and the adsorption layer 160 of the sample are absorbed. The measured values such as the thickness, the adsorption constant, the dissociation constant, and the refractive index are derived. In this case, it is preferable that the operation processor 430 derives a measurement value by obtaining elliptic measurement constants Ψ, Δ regarding the phase difference of the elliptic measurement method in order to improve measurement sensitivity.

The second compensator 440 plays a role of retarding and adjusting the polarization component of the reflected light. The second compensator 440 may be configured to be rotatable or may further include other polarization modulating means.

The spectrometer 450 is used when the light source 310 is white light. This is for spectroscopy of the reflected light and for separating the reflected light having a narrow wavelength range and sending the reflected light to the photodetector 420. In this case, the photodetector 420 may obtain optical data regarding the distribution of reflected light with a two-dimensional image sensor such as a CCD solid-state image pickup device.

[경사 입사구조 프리즘 입사형 실리콘 기반 액침 미세유로 측정방법][Method for Measuring Inclined Structure Prism Incident Type Silicon-Based Liquid Immersion Microfluidic Channel]

Hereinafter, a method and a principle of simultaneous measurement of molecular bonding properties and a buffer solution refractive index will be described with reference to the accompanying drawings.

10 is a flowchart illustrating a method for simultaneously measuring molecular bonding characteristics and a refractive index of a buffer solution according to the present invention. As shown in Figure 10, the measuring method of the present invention is subjected to the first step (S100) to the fifth step (S500).

In the first step (S100), as shown in FIG. 5, the sample injection unit 200 dissolves a sample (not shown) of a biomaterial including a low molecule in the buffer solution 210 to form a microchannel 150 of the microchannel structure 100. ) Will be injected. In this case, the sample injector 100 may inject the buffer solution 210 including the samples having different concentrations in each of the microchannels 150 of the multichannel.

In addition, the buffer solution 210 may be injected with a time difference for each microchannel 150. In addition, the buffer solution 210 may be injected into only some of the microchannels 150, and the remaining microchannels 150 may not be used.

In the second step S200, a sample (not shown) of the biomaterial is adsorbed onto the substrate 120 or the dielectric thin film 130 to form the adsorption layer 160.

On the contrary, the sample has different bonding characteristics by adsorbing the plurality of different self-assembled monolayer films 132 formed on the single channel microchannel structure 100 of FIG. 8 or the plurality of adsorption layers on the same self-assembled monolayer films. An adsorption layer can also be formed.

In the third step S300, the polarizer 320 polarizes predetermined light irradiated from the light source 310 and enters the adsorption layer 160 through the prism 142 of the microchannel structure 100. At this time, the light passes through the incident surface of the prism 142, is refracted at a predetermined angle by the refractive index of the buffer solution 210 existing at the lower end of the prism 142, and then enters the adsorption layer 160. At this time, the polarized incident light has a polarization component of p-wave and s-wave. On the other hand, the incident light should have an incident angle θ that satisfies the p-wave antireflection condition.

In the fourth step S400, the reflected light reflected by the adsorption layer 160 is incident on the polarization detector 400 through the prism 142 of the microchannel structure 100. At this time, the reflected light is an elliptically polarized state.

In the fifth step S500, the polarization detector 400 detects the polarization state of the reflected light. Specifically, the analyzer 410 first receives the elliptically polarized reflected light from the adsorption layer 160 to pass only the light according to the polarization characteristics.

Next, the photodetector 420 obtains predetermined optical data by detecting a change in the polarization component of the reflected light, converts it into an electrical signal, and transmits the converted optical signal to the operation processor 430.

Next, an arithmetic processor 430 with a program using a reflectance measuring method or an ellipsometric method extracts and analyzes optical data converted into an electrical signal, and the adsorption concentration of the sample, the adsorption and dissociation constant, the refractive index, the refractive index of the buffer solution, The same measurement is derived.

At this time, in the present invention, the operation processor 430 obtains the elliptic measurement constant Δ of the phase difference of the elliptic measurement method, measures the refractive index measurement value of the buffer solution, and measures the elliptic measurement constant? The reason is that the elliptic measurement constant Δ of the phase difference under the p-wave antireflection condition is sensitive to the change in the refractive index of the buffer solution and is hardly affected by the bonding characteristics. Therefore, only the change in the refractive index of the buffer solution can be measured. This is because the elliptic measurement constant Ψ regarding the amplitude ratio changes mainly with high sensitivity to the bonding properties of the material.

Therefore, the bonding characteristics of the sample introduced into the buffer solution are measured as Ψ and the refractive index change of the buffer solution including the refractive index that changes when dissolved in the buffer solution or the solvent such as DMSO used to dissolve the sample is measured at the same time as Δ Only the bonding characteristics are obtained.

[실험예]Experimental Example

11 is a view showing the path of the light after reflecting from the sample in the prism vertical incident structure of the prior patent.

In this experiment, the wavelength of the light source 310 is 532 nm, the refractive index of the prism is n = 1.5195 (BK7), and the refraction occurs at the interface between the prism and the buffer solution (n = 1.333), which corresponds to the p-wave antireflection condition. Polarized light enters the sensor on the silicon substrate at 72.15 °.

When the bottom surface of the prism and the silicon substrate are parallel as shown in FIG. 11, the light reflected from the interface between the prism and the buffer solution and the light reflected from the silicon substrate proceed in parallel. In order to minimize the consumption of the sample, if the distance between the prism interface and the silicon substrate is reduced to within about 1 mm, the two lights diffuse as they progress, making it difficult to separate from the photodetector, which is a major factor of measurement error.

However, as shown in FIG. 12A, when the incidence incident structure prism incident silicon-based liquid immersion microfluidic measurement method inclined the bottom of the prism and the silicon substrate is about 2 °, the light is reflected and reflected from the silicon substrate and the light reflected at the interface between the prism and the buffer solution. The light can be completely separated can solve the problems of the prior patent.

In addition, the separated light travels in a direction different from that of the microfluidic structure attached to the prism interface, thereby reducing noise caused by light scattered from the microfluidic structure.

12B illustrates a method for measuring a trapezoidal microfluidic structure and an oblique incidence structure prism incident silicon-based liquid immersion microfluidic channel. When laser light is incident on the narrow microchannel of the trapezoidal microchannel by the cylindrical lens, it is reflected from a 2 ° inclined silicon surface, then passes through the wide microchannel, and then passes through the wide microchannel to act as an aperture. It is possible to minimize noise caused by light scattered irregularly at the microfluidic interface by passing light without scattering the interface of the flow path.

13A and 13B illustrate an apparatus for measuring immersion microchannels according to another embodiment of the present invention. Unlike FIGS. 12A and 12B, the structure of the support 110 is used in the opposite direction in FIGS. 13A and 13B. That is, the embodiment of FIGS. 13A and 13B is configured such that light is incident on the wide microchannel of the trapezoidal microchannel and passes through the narrow microchannel.

In the case of the embodiment of FIGS. 13A and 13B, the scattering may occur more than the structures of FIGS. 12A and 12B, which may cause an error. However, the embodiments of FIGS. 13A and 13B have an advantage that the angle reflected from the bottom of the prism is incident at an angle 2 ° smaller than the angle corresponding to the p-wave antireflection condition, thereby greatly reducing the intensity of the reflected light.

100: fine flow structure

110: support

112: groove

114: first slope

115: second slope

116: through

120: substrate

130: dielectric thin film

132: self-assembled monolayer

140: cover part

142: Prism

143: entrance

144: reflecting surface

146: bulkhead

150: fine euro

152: Inflow path

154: by discharge

160: adsorption layer

200: sample injection part

210: buffer solution

300: polarization generating section

310: light source

320: polarizer

330: collimation lens

340: focusing lens

350: first compensator

400: polarization detection unit

410: The protagonist

420: photodetector

430: operation processor

440: the second compensator

450: Spectrometer

Claims

A microchannel structure including a substrate formed of a support and a semiconductor or a dielectric formed on the support, a prism structure, a cover installed on the support, and a microchannel formed on one of the upper and lower ends of the cover;

A sample injection unit injecting a buffer solution containing a sample of a biomaterial into a micro channel to form an adsorption layer of the sample on a substrate;

A polarization generator for irradiating incident light polarized through the incident surface of the prism to the adsorption layer at an incident angle satisfying a p-wave antireflection condition; And

And a polarization detector configured to receive first reflected light reflected from at least one of the adsorption layer and the substrate through the reflective surface of the prism, and detect a polarization change of the first reflected light.

An inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring device, wherein a surface of the substrate is formed to form a predetermined inclination angle with a bottom surface of the prism.
The method of claim 1,

The first incident light is inclined incident structure prism incident type silicon-based liquid immersion micro-flow measuring apparatus, characterized in that proceed in a different direction from the light reflected from the bottom of the prism.
The method of claim 2,

The polarization detection unit,

An inclined incident structure prism-incident type silicon-based liquid immersion microchannel measuring device, characterized in that for detecting the first reflected light and the light reflected from the bottom of the prism separated.
The method of claim 1,

An inclined incident structure prism incident silicon-based liquid immersion microfluidic measuring device, characterized in that the opening is formed on the upper surface of the support.
The method of claim 4, wherein

The through part is made of a trapezoidal shape,

The trapezoidal shape of the penetrating portion has an inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring device, characterized in that the upper side in the incident surface direction is formed to have a length smaller than the lower side in the reflective surface direction.
The method of claim 5,

The incident light is irradiated to the adsorption layer through the opened through part,

The trapezoidal shape is oblique incident structure prism incident type silicon-based immersion micro-flow measuring apparatus, characterized in that for blocking the reflection of a portion of the incident light.
The method of claim 5,

The upper side of the trapezoidal shape forms a first inclined portion inclined upper side,

The lower side of the trapezoidal shape is characterized in that the inclined incident structure prism incident type silicon-based immersion microfluidic measuring device, characterized in that the inclined second side.
The method of claim 7, wherein

Cross sections of the first inclined portion and the second inclined portion become narrower toward the tip,

An inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring device, characterized in that the tip of the first inclined portion is located below the tip of the second inclined portion.
The method of claim 5,

On the upper side of the trapezoidal upper side of the penetrating portion is formed an inflow passage in which the buffer solution flows into the microchannel,

An inclined incident structure prism-incident type silicon-based liquid immersion microchannel measuring apparatus, characterized in that a discharge path for discharging the buffer solution is formed in the microchannel introduced below the trapezoidal bottom side of the through portion.
The method of claim 1,

And the inclination angle is in a range between 0 seconds and 10 °.
The method of claim 4, wherein

The through part is made of a trapezoidal shape,

The trapezoidal shape of the penetrating portion has an inclined incident structure prism incident type silicon-based liquid immersion microfluidic measuring device, characterized in that the upper side in the incident surface direction is formed to have a length greater than the lower side in the reflective surface direction.
The method of claim 1,

The micro channel structure,

Further comprising a dielectric thin film provided between the substrate and the adsorption layer,

And the first reflected light further includes light reflected from the dielectric thin film.
A microchannel structure having a support and a substrate formed of a semiconductor or a dielectric formed on the support, a flat structure having a cover installed on the support, and a microchannel formed on one of the upper and lower ends of the cover;

A sample injection unit injecting a buffer solution containing a sample of a biomaterial into a micro channel to form an adsorption layer of the sample on a substrate;

A polarization generator for irradiating incident light polarized through the plane of incidence of the flat plate to the adsorption layer at an incidence angle satisfying a p-wave antireflection condition; And

And a polarization detector configured to receive first reflected light reflected from at least one of the adsorption layer and the substrate through the reflective surface of the flat plate, and detect a polarization change of the first reflected light.

And the surface of the substrate is formed to form a predetermined inclination angle with the bottom surface of the flat plate.
support fixture;

A substrate composed of a semiconductor or dielectric formed on a support;

A cover part provided in a prism structure and installed on the support; And

Includes; a micro flow path formed in any one of the upper support and the lower cover portion;

A buffer solution containing a sample of a biomaterial is injected into the microchannel to form an adsorption layer of the sample on the substrate, and the absorption light polarized through the incident surface of the prism is incident at an angle of incidence satisfying a p-wave antireflection condition. Irradiated to the layer, the first reflected light reflected from at least one of the adsorption layer and the substrate is emitted through the reflecting surface of the prism, the surface of the substrate is formed to have a predetermined inclination angle with the bottom surface of the prism A micro flow path structure made of.
support fixture;

A substrate composed of a semiconductor or dielectric formed on a support;

A cover part provided in a flat structure and installed on the support; And

Includes; a micro flow path formed in any one of the upper support and the lower cover portion;

A buffer solution containing a sample of a biomaterial is injected into the microchannel to form an adsorption layer of the sample on the substrate, and the absorption light polarized through the plane of incidence of the plate is incident at an angle of incidence satisfying a p-wave antireflection condition. Irradiated to the layer, the first reflected light reflected from at least one of the adsorption layer and the substrate is emitted through the reflecting surface of the plate, the surface of the substrate is formed to have a predetermined inclination angle with the bottom surface of the plate A micro flow path structure made of.
A first step of injecting a buffer containing a sample of the biomaterial into the microchannel of the microchannel structure;

A second step of adsorbing the sample onto the substrate of the microchannel structure to form an adsorption layer;

A third step of polarizing the light polarizing unit incident light on the adsorption layer through an incident surface of the prism of the microchannel structure at an incident angle satisfying the p-wave antireflection condition;

A fourth step in which first reflected light reflected from at least one of the adsorption layer and the substrate is incident through a reflecting surface of the prism; And

And a fifth step of detecting a polarization change of the first reflected light by the polarization detector.

And a surface of the substrate is formed to form a predetermined inclination angle with a bottom surface of the prism.
A first step of injecting a buffer containing a sample of the biomaterial into the microchannel of the microchannel structure;

A second step of adsorbing the sample onto the substrate of the microchannel structure to form an adsorption layer;

A third step of polarizing the polarizer generating light to be incident on the adsorption layer at an incident angle satisfying a p-wave antireflection condition through an incident surface of the flat plate of the microchannel structure;

A fourth step in which first reflected light reflected from at least one of the adsorption layer and the substrate is incident through a reflecting surface of the plate; And

And a fifth step of detecting a polarization change of the first reflected light by the polarization detector.

And the surface of the substrate is formed to form a predetermined inclination angle with the bottom surface of the flat plate.
The method according to claim 16 or 17,

The first reflected light travels in a different direction from the light reflected from the bottom of the prism,

The method of claim 5, wherein the polarization detector detects the light reflected from the bottom surface of the prism and the first reflected light separately.
The method according to claim 16 or 17,

The fifth step,

Polarizing the first reflected light by an analyzer;

Detecting the polarized first reflected light by a photodetector to obtain predetermined optical data; And

The analysis means obtains an elliptic measurement constant relating to the phase difference of the elliptic measurement method based on the optical data, obtains the refractive index of the buffer solution, obtains an elliptic measurement constant relating to the amplitude ratio, and includes a measured value including the adsorption concentration, adsorption and dissociation constant of the sample. Deriving a; immersion micro-flow measurement method further comprising.